T4噬菌体DNA的复制与调控.ppt

* * T4噬菌体DNA的复制与调控 吴俊 张年辉 卿人韦 T4噬菌体DNA的复制与调控 origin-dependent replication early in infection recombination-dependent replication at later times (RDR) replication mediator protein (RMP)----gp59 and uvsY the sliding clamp-----gp45 Fig.2-1 Model of a replication fork with bacteriophage T4 proteins Fig.1- 1? T4 in vitro system for RDR Features of the RDR pathway First is the strict requirement, under physiological conditions, for an RMP protein(uvsY) to promote the uvsX-catalyzed initiation of leading strand synthesis via the D loop-forming mechanism mentioned above. Second is the requirement for another mediator protein(gp59) to initiate the lagging strand synthesis component of RDR. Third is the intriguing observation, diagrammed in Fig. 1-1, that the synthesis of Okazaki fragments always occurs on the displaced strand of the D loop, and not on the 59 extension of the invading ssDNA. Role of UvsY Protein in Assembly of the T4 Presynaptic Filament UvsY helps uvsX displace gp32 from ssDNA, a reaction necessary for proper formation of the presynaptic filament. UvsY interacts with and stabilizes uvsX-ssDNA filaments after they are assembled. Note: uvsX recombinase cooperatively bound to ssDNA Fig.1-2A Biochemical model for uvsY-mediated assembly of the T4 presynaptic filament 3’ ssDNA tails generated during T4 origin-dependent replication are natural primers for RDR because the presence of homology is guaranteed by the terminal redundancy of T4 DNA .Several other mechanisms exist for 3’ tail generation, including nucleolytic resection of DNA double-stand breaks (DSBs). Both DSB repair and ‘‘normal’’ RDR processes depend on the T4 gp46 and gp47 proteins. The observation of a strong protein–protein interaction between gp46/47 and uvsY raises another intriguing possibility: that nucleolytic resection of DSBs is directly coupled to the assembly of a presynaptic filament on the remaining strand. A hypothetical model for this process is shown in Fig. 1-2B. Hypot